Detection of ethyl glucoronide and ethyl sulfate in Calliphora vicina specimens

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are phase II direct ethanol metabolites used as biomarkers in the diagnosis of acute or chronic alcohol ab/use, depending on the biological matrix evaluated. Because of the postmortem production of alcohol in biological tissues, they have been evaluated as potential biomarkers of ante mortem ethanol ingestion. The purpose of this in vitro study was the evaluation of Calliphora vicina specimens as biological matrix for EtG and EtS. Their presence in entomological species could be useful to discriminate alcohol ingestion in post mortem material. During this in vitro trial, C. vicina was reared using different pig substrates (liver, heart, muscle and tongue). In all trials nine aliquots (30 g) of substrates were used. One milliliter of ethanol was added to three aliquots, while EtG and EtS (1μg/g) were added to other three; three were maintained blank. EtG and EtS were detected using LCMS/MS on the substrates, on the larvae and on the pupae. To assess the presence of EtG and EtS, to study variability caused by substrate, to evaluate the possibility of false positives caused by external alcohol contamination and to estimate the metabolization of ethanol by insects different trials were prepared. EtS was found in all samples where the standard was added, while EtG results were more scattered, because of the high matrix effects observed during the instrumental analysis. A great variation in EtG and EtS analyses was observed in liver, heart and tongue, while muscles appeared the most stable matrix: the three blank aliquots and the three with ethanol did not cause false positives. EtS was found in larvae, pupae and puparia meaning that C. vicina is not able to metabolize either ethanol or EtG and EtS. This study showed that maggots, pupae and puparia could be a useful matrix to evaluate postmortem alcohol ingestion.